The overall objective is to investigate the development of adrenergic and cholinergic junctions made by rat sympathetic principal neurons, in culture. Three related projects are proposed. A major method in each is to grow principal neurons (from superior cervical ganlia rats) one at a time on myocytes, in cultures only about 0.5mm in diameter; we have shown that under these conditions, the neuron makes a high density of junctions on itself and on the target cells. In each project, electrophysiological and electron microscopical methods will be used on the same cell, so that an unambiguous correlation can be made between function and structure. The first project is concerned with the mechanisms which control the choice and expression of transmitter (norepinephrine vs acetylcholine) during development; it arises from our finding that when single neurons are grown on cardiac myocytes for about 2 weeks, some are functionally adrenergic, some cholinergic and some bi-functional (they secrete both norepinephrine and acetylcholine). This and other evidence suggests strongly that all the neurons express adrenergic functions at the outset, but that many are plastic with respect to choice of transmitter, and that under appropriate culture conditions these neurons pass transiently through a condition of dual-function on the way to becoming exclusively cholinergic. At the outset, single neurons grown on cardiac myocytes will be followed with time to demonstrate directly this putative transition from adrenergic to cholinergic functions and to determine its time course. The second project is concerned with a careful comparison of the structure of adrenergic and cholinergic junctions with several target cells, including the neurons themselves, cardiac myocytes and skeletal myotubes. The third project is a parallel study concerned with development of junctions with smooth muscle cells (at the outset, cells from the iris).